[{"id":149836,"pmid":23308053,"pmcid":null,"title":"The RB tumor suppressor positively regulates transcription of the anti-angiogenic protein NOL7.","year":2012,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The expression of the angiogenic phenotype is regulated by a balance of pro-angiogenic and anti-angiogenic factors released into the tumor microenvironment. Nuclear protein 7 (NOL7), a novel tumor suppressor, acts as a master regulator of angiogenesis by downregulating pro-angiogenic factors and upregulating anti-angiogenic factors. Using cervical cancer as a model of investigation, we have previously shown that loss of NOL7 mRNA and protein expression is observed as early as the premalignant phase. Analysis of the gene failed to identify tumor-specific promoter methylation or coding region mutations, suggesting that NOL7 loss may be mediated by aberrant expression of its upstream regulators. In this study, we show that the RB tumor suppressor gene (RB) positively regulates NOL7 at the transcriptional level by recruiting transcription factors and transcription machinery proteins to its promoter region. Conversely, the loss of RB represses NOL7 transcription by inhibiting assembly of these proteins. This loss of NOL7 expression is also observed in RB-deficient human malignancies. Together, this work further characterizes the transcriptional activator function of RB and defines a potential role for RB in regulating angiogenesis through activation of NOL7. Current anti-angiogenic therapies lack long-term efficacy, as they are unable to target the diverse angiogenic signals generated by tumors. Our data provide evidence to support the hypothesis that reactivation of pRB can potentially modulate the expression of the angiogenic phenotype through regulation of NOL7. Therefore, this knowledge may be employed to design more comprehensive and effective therapies.","journal":null,"figures":[],"_authors":null},{"id":23,"pmid":23186163,"pmcid":null,"title":"Toward a comprehensive characterization of a human cancer cell phosphoproteome.","year":2013,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.","journal":null,"figures":[],"_authors":null},{"id":149835,"pmid":23085760,"pmcid":null,"title":"The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression.","year":2013,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specifically interacts with TSP-1 mRNA through its 3'UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 3'UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 significantly alters TSP-1 expression and may be a master regulator that coordinates the post-transcriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype.","journal":null,"figures":[],"_authors":null},{"id":1445,"pmid":22658674,"pmcid":null,"title":"Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.","year":2012,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed \"interactome capture,\" to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.","journal":null,"figures":[],"_authors":null},{"id":149834,"pmid":22123719,"pmcid":null,"title":"Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.","year":2012,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.","journal":null,"figures":[],"_authors":null},{"id":149833,"pmid":21818416,"pmcid":null,"title":"The dynamics of the alternatively spliced NOL7 gene products and role in nucleolar architecture.","year":null,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Three alternatively spliced forms of the human NOL7 gene coding for relatively small proteins were identified. The two shorter forms were generated by intron retention events, and each isoform was differently localized within the cell. The NOL7-SP1 long form (29 kD) localized to the nucleolus, SP2 was nucleoplasmic, while SP3 was distributed throughout the whole cell. NOL7-SP1 was confined to the nucleolar granular component, and during cell division disassociated from the nucleolus. Knockdown of NOL7-SP1 levels abrogated nucleolar architecture, in particular the internal regions, and reduced cell proliferation. Analysis of the nucleolar dynamics of the SP1 protein during interphase showed nucleolar high binding affinity. Dissection of protein domains showed that nucleolar targeting was mediated by a unique C-terminal nucleolar localization sequence (NoLS). However, this sequence was not sufficient for conferring high binding affinity, which required additional regions of the protein. Our analysis shows that NOL7 is important for maintaining internal nucleolar structure and cell growth rates, and that while specific protein localization can be obtained by specific short localization motifs, nucleolar residency through binding must be mediated by a synergistic combination of protein modules.","journal":null,"figures":[],"_authors":null},{"id":149832,"pmid":20875127,"pmcid":null,"title":"Identification and functional analysis of NOL7 nuclear and nucleolar localization signals.","year":2010,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated.","journal":null,"figures":[],"_authors":null},{"id":149831,"pmid":20206243,"pmcid":null,"title":"Identification and characterization of the human NOL7 gene promoter.","year":2010,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"NOL7 is a candidate tumor suppressor gene that localizes to 6p23, a chromosomal region frequently associated with loss of heterozygosity in a number of malignancies including cervical cancer (CC). Re-expression of NOL7 in CC cells suppresses in vivo tumor growth by 95% and alters the angiogenic phenotype by modulating the expression of VEGF and TSP1. Here, we describe the determination of two NOL7 transcriptional start sites (TSS), the cloning of its regulatory promoter region, and the identification of transcription factors that regulate its expression. Using 5' Rapid amplification of complementary DNA ends (RACE), two transcriptional start sites were identified. Deletion analysis determined that the essential elements required for the optimal promoter activity of NOL7 were 560 bp upstream of its translation start site. In silico analysis suggested that the promoter region contained potential binding sites for the SP1, c-Myc and RXRalpha transcription factors as well as an overall GC content of greater than 60%. Chromatin immunoprecipitation (ChIP) confirmed that SP1, c-Myc and RXRalpha bound to the NOL7 promoter region. Finally, we demonstrate that NOL7 expression was positively regulated by c-Myc and RXRalpha. These results demonstrate that the NOL7 promoter region possesses each of the key elements of a TATA-less promoter. In addition, the positive regulation of NOL7 by c-Myc and RXRalpha provides additional mechanistic insights into the potential role of NOL7 in CC and other malignancies.","journal":null,"figures":[],"_authors":null},{"id":41,"pmid":18029348,"pmcid":null,"title":"Toward a confocal subcellular atlas of the human proteome.","year":2008,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.","journal":null,"figures":[],"_authors":null},{"id":638,"pmid":17974005,"pmcid":null,"title":"The full-ORF clone resource of the German cDNA Consortium.","year":2007,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.","journal":null,"figures":[],"_authors":null},{"id":144,"pmid":17207965,"pmcid":null,"title":"hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.","year":2007,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at ). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.","journal":null,"figures":[],"_authors":null},{"id":149830,"pmid":16205646,"pmcid":null,"title":"NOL7 is a nucleolar candidate tumor suppressor gene in cervical cancer that modulates the angiogenic phenotype.","year":2006,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Cervical cancer is associated with human papilloma virus infection. However, this infection is insufficient to induce transformation and progression. Loss of heterozygosity analyses suggest the presence of a tumor suppressor gene (TSG) on chromosome 6p21.3-p25. Here we report the cloning NOL7, its mapping to chromosome band 6p23, and localization of the protein to the nucleolus. Fluorescence in situ hybridization analysis demonstrated an allelic loss of an NOL7 in cultured tumor cells and human tumor samples. Transfection of NOL7 into cervical carcinoma cells inhibited their growth in mouse xenografts, confirming its in vivo tumor suppressor activity. The induction of tumor dormancy correlated with an angiogenic switch caused by a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These data suggest that NOL7 may function as a TSG in part by modulating the expression of the angiogenic phenotype.","journal":null,"figures":[],"_authors":null},{"id":3108,"pmid":15635413,"pmcid":null,"title":"Nucleolar proteome dynamics.","year":2005,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.","journal":null,"figures":[],"_authors":null},{"id":4,"pmid":15489334,"pmcid":null,"title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","year":2004,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.","journal":null,"figures":[],"_authors":null},{"id":3,"pmid":14702039,"pmcid":null,"title":"Complete sequencing and characterization of 21,243 full-length human cDNAs.","year":2004,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"As a base for human transcriptome and functional genomics, we created the \"full-length long Japan\" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.","journal":null,"figures":[],"_authors":null},{"id":86,"pmid":14574404,"pmcid":null,"title":"The DNA sequence and analysis of human chromosome 6.","year":2003,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.","journal":null,"figures":[],"_authors":null},{"id":2,"pmid":12477932,"pmcid":null,"title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).","journal":null,"figures":[],"_authors":null},{"id":3273,"pmid":12429849,"pmcid":null,"title":"Functional proteomic analysis of human nucleolus.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The notion of a \"plurifunctional\" nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of approximately 350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.","journal":null,"figures":[],"_authors":null},{"id":4317,"pmid":11790298,"pmcid":null,"title":"Directed proteomic analysis of the human nucleolus.","year":2002,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized.","journal":null,"figures":[],"_authors":null},{"id":71106,"pmid":9875212,"pmcid":null,"title":"Polar amino acid-rich sequences bind to polyglutamine tracts.","year":1998,"pages":null,"doi":null,"keywords":[],"mesh":[],"abstractText":"Polyglutamine tracts are found in different proteins including transcription factors and cofactors as well as in triplet repeat disease gene products. To characterize the protein motif that binds to the polyglutamine tract, we screened a human embryonic brain cDNA library with the polyglutamine tract of Brn-2 as bait using the yeast two-hybrid method. All six isolated clones encoding polyglutamine tract binding proteins were rich in polar amino acids. Three of these clones could form polar helical structures. These observations suggest that polar amino acid-rich sequences are essential for binding to the polyglutamine tract.","journal":null,"figures":[],"_authors":null}]